PROJECT SUMMARY While immunotherapy is a breakthrough in our fight against cancer, only a subset of patients display robust and long-lasting responses highlighting the critical need for novel approaches to be used alone or in combination with current therapeutic regimens. TH17 cells, which express the lineage defining transcription factor RORgt, have emerged as targets to enhance antitumor immunity, with RORg agonists currently in Phase 1/2 clinical trials. The transcriptional repressors REV-ERBa and REV-ERBb are often co-expressed in the same tissues as RORg(t) and bind the same DNA response elements, which suggests mutual cross talk and co-regulation of their target genes. Our preliminary data demonstrates that the REV-ERBs are ligand-regulated transcription factors and play critical roles in TH17 responses, including regulation of IL-17A expression through competition with RORgt at their shared DNA consensus sequence(s), and regulation of RORgt expression itself. We have generated small molecule ligands that enhance the REV-ERBs repressive function and inhibit TH17 cell development in vitro and in vivo. Thus, we hypothesize that identification of ligands that inhibit the REV-ERBs repressive activity will enhance TH17 responses and antitumor immunity. In order to identify ligands that inhibit REV-ERB activity, we have designed a high-throughput screening (HTS) compatible primary assay that detects direct ligand binding to each receptor. Our goal is to implement a full HTS-campaign using the Scripps Institutional Drug Discovery Library (SDDL) to identify, validate, and characterize potent small molecule inverse agonists of REV-ERB activity. To achieve our goal, we will miniaturize our assay into a 1,536-well plate format and upon meeting HTS automation criteria, carry out a ?full-deck? HTS campaign to screen the >640,000 compounds in the SDDL (Aim 1). Cheminformatic analysis of ?hits? will help identify the most promising leads by structural clustering, bioinformatics analysis of compounds to determine promiscuity, and scaffold analysis to determine ease of chemical synthesis and tractability for further medicinal chemistry efforts to perform structure- activity relationship studies. In Aim 2, we will use a cascade of follow up assays to validate screening hits, determine specificity, and begin to understand mechanism of action of REV-ERB-mediated transcriptional activity. Finally, in Aim 3, validated HTS hits will be advanced to early medicinal chemistry for lead optimization and characterization of novel negative regulators of REV-ERBa/b. We expect that completion of this application will deliver multiple structurally distinct REV-ERB inverse agonists that exhibit suitable levels of cellular activity, potency, and selectivity. Our collaborative research team has a strong track record of performing high-throughput screens, selective optimization of scaffolds, and in vitro and in vivo characterization of compounds. Collectively, our screening approach puts us in a unique position to identify, validate, and characterize novel REV-ERB- selective small molecule inverse agonists for the study of the REV-ERB?s function in enhancing TH17 responses and to determine whether targeting these receptors is a viable option for immunotherapy.